The objective of this project is to construct an effective in vivo and in vitro gene transfer system by utilizing the highly efficient endocytosis process whereby asialoglycoproteins are taken up by normal hepatocytes. This should enable us to examine the biological effects, particularly with respect to oncogenesis, of introducing specific genes, both under in vivo and in vitro conditions into a normal highly differentiated cell. This is accomplished by covalently coupling the asialoglycoprotein to the DNA by using two reagents N-acetyl-N'-(p-glyoxylbenzoyl)cystamine and 2-iminothiolane. The former reacts specifically with nonpaired guanine residues and upon reduction generates a free sulfhydryl group. The latter reacts with the protein to provide another sulfhydryl group which is subsequently conjugated to DNA by an intermolecular disulfide interchange reaction. The experimental model currently under study is the rat liver. The initial coupling has been done using two transformation specific viral DNAs, namely the bovine papillomavirus DNA and the cDNA clone of the Harvey RNA tumor virus. Both tumor viruses have been well characterized in terms of transforming ability. The bovine papillomavirus DNA was prepared from pBR322 recombinants and tailed with approximately 50 residues of dGTP using terminal deoxytransferase. G-tailed viral DNA was coupled to modified asialofetuin by incubation under appropriate conditions. Harvey cDNA is presently being prepared and modified in a similar manner. Uptake and expression of viral DNAs coupled with asialofetuin into primary rat hepatocytes is currently being determined.